Project Summary/Abstract Understanding why disease progression in the majority of patients with Amyotrophic Lateral Sclerosis (ALS) occurs in contiguous anatomic regions over time is one of the fundamental limitations to designing disease modifying therapies that can be utilized after a diagnosis. Several studies in ALS rodent models suggest that astrocytes play a role in disease propagation after onset. However, astrocyte dysfunction is not an observation merely limited to ALS rodent models but, importantly, has been one of the most consistent observations in humans with ALS when examined in situ as well as using human cells in vitro. Astrocytes form a highly coupled intercellular network in the central nervous system (CNS) through gap junctions (GJs) and hemichannels (HC) composed of 6 connexin subunits arranged around a central pore. Connexins in astrocytes have key roles: homeostatic buffering, synchronization of astrocyte networks, metabolic support for neurons, and regulation of vascular physiology. They can also propagate Ca2+ waves and modulate synaptic events or release gliotransmitters, including glutamate and ATP, through hemichannels. Connexin 43 (Cx43) is the predominant connexin in astrocytes and is expressed ubiquitously in the CNS. Our recently published studies have demonstrated that astrocyte expression of Cx43 is increased in the frontal cortex and spinal cords of ALS patients, an observation mirrored in the SOD1 mouse model of ALS. This phenomenon is not merely a non-specific effect of reactive astrocytosis as we were also able to show, using in vitro and in vivo mouse modeling, that SOD1 astrocyte-mediated motor neuron toxicity was, at least in part, mediated through Cx43 hemichannels. This proposal builds upon these initial observations by using a fully humanized, spinal cord-specific, ALS iPSC-astrocyte/motor neuron platform to investigate Cx43 HC localization at the hiPSC-astrocyte membrane, examine mechanisms by which Cx43 HC opening is modulated in the context of ALS, and understand how this affects Cx43 HC-mediated ALS hiPSC-astrocyte toxicity to motor neurons. Using newly reported specific blockers of HC activity we will now be able to dissect the specific contributions of Cx43 HC to motor neuron death. As we think about translational potential, we will test these Cx43 HC blockers that can penetrate the blood brain barrier, are well tolerated and orally available, for efficacy in human iSPC-derived astrocyte mediated MN toxicity platforms and in two ALS rodent models. What we learn from this proposal will allow us to develop a systematic approach to ALS therapeutics that combines human tissue data with novel in vitro modeling, and finally to in vivo translational applications.